Functional Studies Of Membrane Proteins Research Articles

A nondestructive membrane engineering method using an amphiphilic polymer.

The cellular signaling process or ion transport is mediated by membrane proteins (MPs) located on the cell surface, and functional studies of MPs have mainly been conducted using cells endogenously or transiently expressing target proteins. Reconstitution of purified MPs in the surface of live cells would have advantages of short manipulation time and ability to target cells in which gene transfection is difficult. However, direct reconstitution of MPs in live cells has not been established. The traditional detergent-mediated reconstitution method of MPs into a lipid bilayer cannot be applied to live cells because this disrupts and reforms the lipid bilayer structure, which is detrimental to cell viability. In this study, we demonstrated that GPCRs (prostaglandin E2 receptor 4 [EP4] and glucagon-like peptide-1 receptor [GLP1R]) or serotonin receptor 3A (5HT3A), a ligand-gated ion channel, stabilized with amphiphilic poly-γ-glutamate (APG), can be reconstituted into mammalian cell plasma membranes without affecting cell viability. Furthermore, 5HT3A reconstituted in mammalian cells showed ligand-dependent Ca2+ ion transport activity. APG-mediated reconstitution of GPCR in synthetic liposomes showed that electrostatic interaction between APG and membrane surface charge contributed to the reconstitution process. This APG-mediated membrane engineering method could be applied to the functional modification of cell membranes with MPs in live cells.

Lipid Bilayer Reformation Using the Wiping Blade for Improved Ion Channel Analysis.

The measurement of ion permeation activity across planar lipid bilayers is a useful technique for the functional analysis and drug evaluation of ion channels at the single-molecule level. To enhance the data throughput, parallelization of lipid bilayers is desirable. However, existing parallelized approaches face challenges in simultaneously and efficiently measuring ion channel activities under various conditions on one chip. In this study, we propose an approach to overcome these limitations by developing a device capable of repeated measurements of ion channels incorporated into individually arrayed lipid bilayers. Our device forms an array of a lipid bilayer at a micropore on a separator by merging two lipid monolayers assembled on the surface of aqueous droplets. We introduce a vertically moving, blade-shaped module─referred to as a "wiping blade"─which enables controlled disruption and reformation of the bilayer at the micropore. By optimizing the surface properties and clearance of the wiping blade, we successfully achieved repeated bilayer formation. The arrayed lipid bilayer device with the integrated wiping blade module demonstrates a 5-fold improvement in data throughput during ion channel activity measurements. Finally, we validate the practical utility of our device by evaluating the effects of an ion channel inhibitor. The developed device opens new avenues for high-throughput analysis and screening of ion channels, leading to significant advancements in drug discovery and functional studies of membrane proteins. It offers a powerful tool for researchers in the field and holds promise for accelerating drug development by targeting ion channels.

Sulfonated polystyrenes: pH and Mg2+-insensitive amphiphilic copolymers for detergent-free membrane protein isolation

Amphiphilic polymers are increasingly applied in the detergent-free isolation and functional studies of membrane proteins. However, the carboxylate group present in the structure of many popular variants, such as styrene-maleic acid (SMA) copolymers, brings limitations in terms of polymer sensitivity to precipitation at acidic pH or in the presence of divalent metal cations. Herein, we addressed this problem by replacing carboxylate with the more acidic sulfonate groups. To this end, we synthesized a library of amphiphilic poly[styrene-co-(sodium 4-styrene sulfonate)] copolymers (termed SSS), differing in their molecular weight and overall polarity. Using model cell membranes (Jurkat), we identified two copolymer compositions (SSS-L30 and SSS-L36) that solubilized membranes to an extent similar to SMA. Interestingly, the density gradient ultracentrifugation/SDS-PAGE/Western blotting analysis of cell lysates revealed a distribution of studied membrane proteins in the gradient fractions that was different than for SMA-solubilized membranes. Importantly, unlike SMA, the SSS copolymers remained soluble at low pH and in the presence of Mg2+ ions. Additionally, the solubilization of DMPC liposomes by the lead materials was studied by turbidimetry, DLS, SEC, and high-resolution NMR, revealing, for SSS-L36, the formation of stable particles (nanodiscs), facilitated by the direct hydrophobic interaction of the copolymer phenyls with lipid acyl chains.

The Advanced Properties of Circularized MSP Nanodiscs Facilitate High-resolution NMR Studies of Membrane Proteins

Membrane mimetics are essential for structural and functional studies of membrane proteins. A promising lipid-based system are phospholipid nanodiscs, where two copies of a so-called membrane scaffold protein (MSP) wrap around a patch of lipid bilayer. Consequently, the size of a nanodisc is determined by the length of the MSP. Furthermore, covalent MSP circularization was reported to improve nanodisc stability. However, a more detailed comparative analysis of the biophysical properties of circularized and linear MSP nanodiscs for their use in high-resolution NMR has not been conducted so far. Here, we analyze the membrane fluidity and temperature-dependent size variability of circularized and linear nanodiscs using a large set of analytical methods. We show that MSP circularization does not alter the membrane fluidity in nanodiscs. Further, we show that the phase transition temperature increases for circularized versions, while the cooperativity decreases. We demonstrate that circularized nanodiscs keep a constant size over a large temperature range, in contrast to their linear MSP counterparts. Due to this size stability, circularized nanodiscs are beneficial for high-resolution NMR studies of membrane proteins at elevated temperatures. Despite their slightly larger size as compared to linear nanodiscs, 3D NMR experiments of the voltage-dependent anion channel 1 (VDAC1) in circularized nanodiscs have a markedly improved spectral quality in comparison to VDAC1 incorporated into linear nanodiscs of a similar size. This study provides evidence that circularized MSP nanodiscs are a promising tool to facilitate high-resolution NMR studies of larger and challenging membrane proteins in a native lipid environment.

Detergent-Free Isolation of Membrane Proteins and Strategies to Study Them in a Near-Native Membrane Environment.

Atomic-resolution structural studies of membrane-associated proteins and peptides in a membrane environment are important to fully understand their biological function and the roles played by them in the pathology of many diseases. However, the complexity of the cell membrane has severely limited the application of commonly used biophysical and biochemical techniques. Recent advancements in NMR spectroscopy and cryoEM approaches and the development of novel membrane mimetics have overcome some of the major challenges in this area. For example, the development of a variety of lipid-nanodiscs has enabled stable reconstitution and structural and functional studies of membrane proteins. In particular, the ability of synthetic amphipathic polymers to isolate membrane proteins directly from the cell membrane, along with the associated membrane components such as lipids, without the use of a detergent, has opened new avenues to study the structure and function of membrane proteins using a variety of biophysical and biological approaches. This review article is focused on covering the various polymers and approaches developed and their applications for the functional reconstitution and structural investigation of membrane proteins. The unique advantages and limitations of the use of synthetic polymers are also discussed.

Membrane contact probability: An essential and predictive character for the structural and functional studies of membrane proteins.

One of the unique traits of membrane proteins is that a significant fraction of their hydrophobic amino acids is exposed to the hydrophobic core of lipid bilayers rather than being embedded in the protein interior, which is often not explicitly considered in the protein structure and function predictions. Here, we propose a characteristic and predictive quantity, the membrane contact probability (MCP), to describe the likelihood of the amino acids of a given sequence being in direct contact with the acyl chains of lipid molecules. We show that MCP is complementary to solvent accessibility in characterizing the outer surface of membrane proteins, and it can be predicted for any given sequence with a machine learning-based method by utilizing a training dataset extracted from MemProtMD, a database generated from molecular dynamics simulations for the membrane proteins with a known structure. As the first of many potential applications, we demonstrate that MCP can be used to systematically improve the prediction precision of the protein contact maps and structures.

Measurement of Residual Dipolar Couplings Using Magnetically Aligned and Flipped Nanodiscs.

Recent developments in lipid nanodisc technology have successfully overcome the major challenges in the structural and functional studies of membrane proteins and drug delivery. Among the different types of nanodiscs, the use of synthetic amphiphilic polymers created new directions including the applications of solution and solid-state NMR spectroscopy. The ability to magnetically align large-size (>20 nm diameter) polymer nanodiscs and flip them using paramagnetic lanthanide ions has enabled high-resolution studies on membrane proteins using solid-state NMR techniques. The use of polymer-based macro-nanodiscs (>20 nm diameter) as an alignment medium to measure residual dipolar couplings (RDCs) and residual quadrupole couplings by NMR experiments has also been demonstrated. In this study, we demonstrate the use of magnetically aligned and 90°-flipped polymer nanodiscs as alignment media for structural studies on proteins by solution NMR spectroscopy. These macro-nanodiscs, composed of negatively charged SMA-EA polymers and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids, were used to measure residual 1H-15N dipolar couplings (RDCs) from the water-soluble ∼21 kDa uniformly 15N-labeled flavin mononucleotide binding domain (FBD) of cytochrome-P450 reductase. The experimentally measured 1H-15N RDC values are compared with the values calculated from the crystal structures of cytochrome-P450 reductase that lacks the transmembrane domain. The N-H RDCs measured using aligned and 90°-flipped nanodiscs show a modulation by the function (3 cos2 θ - 1), where θ is the angle between the N-H bond vector and the applied magnetic field direction. This successful demonstration of the use of two orthogonally oriented alignment media should enable structural studies on a variety of systems including small molecules, DNA, and RNA.

Evaluation of DIBMA nanoparticles of variable size and anionic lipid content as tools for the structural and functional study of membrane proteins

Amphiphilic maleic acid-containing polymers allow for the direct extraction of membrane proteins into stable, homogenous, water-soluble copolymer/lipid nanoparticles without the use of detergents. By adjusting the polymer/lipid ratio, the size of the nanoparticles can be tuned at convenience for the incorporation of protein complexes of different size. However, an increase in the size of the lipid nanoparticles may correlate with increased sample heterogeneity, thus hampering their application to spectroscopic and structural techniques where highly homogeneous samples are desirable. In addition, size homogeneity can be affected by low liposome solubilization efficiency by DIBMA, which carries a negative charge, in the presence of high lipid charge density.In this work, we apply biophysical tools to characterize the size and size heterogeneity of large (above 15 nm) lipid nanoparticles encased by the diisobutylene/maleic acid (DIBMA) copolymer at different DIBMA/lipid ratios and percentages of anionic lipids. Importantly, for nanoparticle preparations in the diameter range of 40 nm or below, the size homogeneity of the DIBMA/lipid nanoparticles (DIBMALPs) remains unchanged. In addition, we show that anionic lipids do not affect the production, size and size homogeneity of DIBMALPs. Furthermore, they do not affect the overall lipid dynamics in the membrane, and preserve the functionality of an enclosed membrane protein.This work strengthens the suitability of DIBMALPs as universal, native-like lipid environments for functional studies of membrane proteins and provide useful insight on the suitability of these systems for those structural techniques requiring highly homogeneous sample preparations.

Benchmarks of SMA-Copolymer Derivatives and Nanodisc Integrity.

Poly(styrene-co-maleic acid) or SMA and its derivatives, a family of synthetic amphipathic copolymers, are increasingly used to directly solubilize cell membranes to functionally reconstitute membrane proteins in native-like copolymer-lipid nanodiscs. Although these copolymers act, de facto, like a "macromolecular detergent", the polymer-based lipid-nanodiscs has been demonstrated to be an excellent membrane mimetic for structural and functional studies of membrane proteins and their complexes by a variety of biophysical and biochemical approaches. In many studies reported in the literature, the choice of the right SMA formulation can depend on a number of factors, and the experimental conditions are typically developed according to a trial-and-error process since each studied system requires adapted protocols. While increasing number of nanodisc-forming copolymers are reported to be useful and they provide flexibilities in optimizing the sample preparation conditions, it is important to develop a systematic protocol that can be used for various applications. In this context, there is a vital necessity of benchmarking the performances of existing copolymer formulations, assessing crucial parameters for the successful extraction, isolation, and stabilization of membrane proteins. In this study, we compare both copolymers and copolymer-lipid nanodiscs obtained by SMA-EA with a set of anionic XIRAN copolymer formulations commercially available under the names of SL25010 P, SL30010 P, and SL40005 P. The reported results show how the critical micellar concentration (c.m.c.) of each copolymer is significantly altered in the presence of lipids and confirms the existence of an equilibrium between nanodisc-bound and "free" or "micellar" copolymer chains in the solution. We believe that these findings can be exploited to optimize studies that involve the necessity of special copolymers, which would not only simplify the applications but also broaden the scope of polymer-based nanodiscs.

Current problems and future avenues in proteoliposome research.

Membrane proteins (MPs) are the gatekeepers between different biological compartments separated by lipid bilayers. Being receptors, channels, transporters, or primary pumps, they fulfill a wide variety of cellular functions and their importance is reflected in the increasing number of drugs that target MPs. Functional studies of MPs within a native cellular context, however, is difficult due to the innate complexity of the densely packed membranes. Over the past decades, detergent-based extraction and purification of MPs and their reconstitution into lipid mimetic systems has been a very powerful tool to simplify the experimental system. In this review, we focus on proteoliposomes that have become an indispensable experimental system for enzymes with a vectorial function, including many of the here described energy transducing MPs. We first address long standing questions on the difficulty of successful reconstitution and controlled orientation of MPs into liposomes. A special emphasis is given on coreconstitution of several MPs into the same bilayer. Second, we discuss recent progress in the development of fluorescent dyes that offer sensitive detection with high temporal resolution. Finally, we briefly cover the use of giant unilamellar vesicles for the investigation of complex enzymatic cascades, a very promising experimental tool considering our increasing knowledge of the interplay of different cellular components.

1,3,5-Triazine-Cored Maltoside Amphiphiles for Membrane Protein Extraction and Stabilization.

Despite their major biological and pharmacological significance, the structural and functional study of membrane proteins remains a significant challenge. A main issue is the isolation of these proteins in a stable and functional state from native lipid membranes. Detergents are amphiphilic compounds widely used to extract membrane proteins from the native membranes and maintain them in a stable form during downstream analysis. However, due to limitations of conventional detergents, it is essential to develop novel amphiphiles with optimal properties for protein stability in order to advance membrane protein research. Here we designed and synthesized 1,3,5-triazine-cored dimaltoside amphiphiles derived from cyanuric chloride. By introducing variations in the alkyl chain linkage (ether/thioether) and an amine-functionalized diol linker (serinol/diethanolamine), we prepared two sets of 1,3,5-triazine-based detergents. When tested with several model membrane proteins, these agents showed remarkable efficacy in stabilizing three transporters and two G protein-coupled receptors. Detergent behavior substantially varied depending on the detergent structural variation, allowing us to explore detergent structure-property-efficacy relationships. The 1,3,5-triazine-based detergents introduced here have significant potential for membrane protein study as a consequence of their structural diversity and universal stabilization efficacy for several membrane proteins.

Cryo-EM sample preparation method for extremely low concentration liposomes

Liposomes are widely used as delivery systems in pharmaceutical, cosmetics and food industries, as well as a system for structural and functional study of membrane proteins. To accurately characterize liposomes, cryo-Electron Microscopy (cryo-EM) has been employed as it is the most precise and direct method to determine liposome lamellarity, size, shape and ultrastructure. However, its use is limited by the number of liposomes that can be trapped in the thin layer of ice that spans holes in the perforated carbon film on EM grids. We report a long-incubation method for increasing the density of liposomes in holes. By increasing the incubation time, high liposome density was achieved even with extremely dilute (in the nanomolar range) liposome solutions. This long-incubation method has been successfully employed to study the structure of an ion channel reconstituted into liposomes.

Detergent-free solubilization of human Kv channels expressed in mammalian cells.

Styrene-maleic acid (SMA) copolymers are used to extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding SMA lipid particles (SMALPs). SMALPs can serve as stable water-soluble nanocontainers for structural and functional studies of membrane proteins. Here, we used SMA copolymers to study full-length pore-forming α-subunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an α-subunit hKCNQ1 and its regulatory transmembrane KCNE1 β-subunit (hKCNE1-hKCNQ1) with added affinity tags, expressed in mammalian COS-1 cells. All these recombinant proteins were shown to be functionally active. Treatment with the SMA copolymer, followed by purification on the affinity column, enabled extraction of all three channels. A DLS experiment demonstrated that negative stain electron microscopy and single particle image analysis revealed a four-fold symmetry within channel-containing SMALPs, which indicates that purified hKCNH5 and hKCNQ1 channels, as well as the hKCNE1-hKCNQ1 fusion construct, retained their structural integrity as tetramers.

Towards unidirectional reconstitution of membrane proteins into liposomes

Functional studies of membrane proteins (MP) in their native environment (the cellular membrane) is challenging due to the complexity of the native membranes and measurement interferences from other membrane constituents or reactions. Therefore, MPs are often purified and integrated into systems which mimic their natural environment in a membrane as e.g. liposomes or nanodiscs, a process called reconstitution, allowing to investigate its function and structural aspects without any disturbing background. The most important problem during MP-reconstitution is the often random orientation of the MP in the liposomal membrane after reconstitution. For functional studies of the MP of interest and quantitative analysis of its properties, unidirectional orientation in the liposomal membrane is required. Previous work of other groups, where MPs have been reconstituted and studied, did not include a final and universal approach (Rigaud JL, Levy D. (2003) Methods Enzymol. 372:65-86.; Tunuguntla R et al. (2013) Biophys. J. 105(6):1388-96.) and procedures have to be individually optimized for an enrichment of enzyme orientation. In most cases, however, orientation cannot be influenced and is thought to depend on the three-dimensional structure of the protein (Tunuguntla R et al. (2013) Biophys. J. 105(6):1388-96; Nordlund G et al. (2014) Nat. Commun. 5:4303.). We are currently developing and establishing a universal method to force unidirectional reconstitution of MPs by the aid of a molecular unit that can be attached to every protein. Our method is based on the observation that the F1F0 ATP synthase with its large hydrophilic head piece (F1 part) orients highly uniformly when embedded in preformed vesicles. We try to mimic this situation with a bioengineering approach that should be applicable to a wide variety of proteins. Once such a method is fully established, the interplay of two or more membrane proteins can be investigated more quantitatively.

Nanodisc-Forming Scaffold Protein Promoted Retardation of Amyloid-Beta Aggregation

Peptidic nanodiscs are useful membrane mimetic tools for structural and functional studies of membrane proteins, and membrane interacting peptides including amyloids. Here, we demonstrate anti-amyloidogenic activities of a nanodisc-forming 18-residue peptide (denoted as 4F), both in lipid-bound and lipid-free states by using Alzheimer's amyloid-beta (Aβ40) peptide as an example. Fluorescence-based amyloid fibrillation kinetic assays showed a significant delay in Aβ40 amyloid aggregation by the 4F peptide. In addition, 4F-encased lipid nanodiscs, at an optimal concentration of 4F (>20 μM) and nanodisc size (<10 nm), significantly affect amyloid fibrillation. A comparison of experimental results obtained from nanodiscs with that obtained from liposomes revealed a substantial inhibitory efficacy of 4F-lipid nanodiscs against Aβ40 aggregation and were also found to be suitable to trap Aβ40 intermediates. A combination of atomistic molecular dynamics simulations with NMR and circular dichroism experimental results exhibited a substantial change in Aβ40 conformation upon 4F binding through electrostatic and π–π interactions. Specifically, the 4F peptide was found to interfere with the central β-sheet-forming residues of Aβ40 through substantial hydrogen, π–π, and π–alkyl interactions. Fluorescence experiments and coarse-grained molecular dynamics simulations showed the formation of a ternary complex, where Aβ40 binds to the proximity of peptidic belt and membrane surface that deaccelerate amyloid fibrillation. Electron microscopy images revealed short and thick amyloid fibers of Aβ40 formed in the presence of 4F or 4F-lipid nanodsics. These findings could aid in the development of amyloid inhibitors as well as in stabilizing Aβ40 intermediates for high-resolution structural and neurobiological studies.

Macrodiscs Comprising SMALPs for Oriented Sample Solid-State NMR Spectroscopy of Membrane Proteins

Macrodiscs, which are magnetically alignable lipid bilayer discs with diameters of >30 nm, were obtained by solubilizing protein-containing liposomes with styrene-maleic acid copolymers. Macrodiscs provide a detergent-free phospholipid bilayer environment for biophysical and functional studies of membrane proteins under physiological conditions. The narrow resonance linewidths observed from membrane proteins in styrene-maleic acid macrodiscs advance structure determination by oriented sample solid-state NMR spectroscopy.

An Engineered Lithocholate-Based Facial Amphiphile Stabilizes Membrane Proteins: Assessing the Impact of Detergent Customizability on Protein Stability.

Amphiphiles are critical tools for the structural and functional study of membrane proteins. Membrane proteins encapsulated by conventional head-to-tail detergents tend to undergo structural degradation, necessitating the development of structurally novel agents with improved efficacy. In recent years, facial amphiphiles have yielded encouraging results in terms of membrane protein stability. Herein, we report a new facial detergent (i.e., LFA-C4) that confers greater stability to tested membrane proteins than the bola form analogue. Owing to the increased facial property and the adaptability of the detergent micelles in complex with different membrane proteins, LFA-C4 yields increased stability compared to n-dodecyl-β-d-maltoside (DDM). Thus, this study not only describes a novel maltoside detergent with enhanced protein-stabilizing properties, but also shows that the customizable nature of a detergent plays an important role in the stabilization of membrane proteins. Owing to both synthetic convenience and enhanced stabilization efficacy for a range of membrane proteins, the new agent has major potential in membrane protein research.

Real-Time Monitoring of Lipid Exchange via Fusion of Peptide Based Lipid-Nanodiscs

Lipid bilayer nanodiscs serve as excellent membrane mimetics for structural and functional studies of mem-brane proteins. Peptide-based lipid nanodiscs are unique in rendering the flexibility in size and exchange of membrane components. While the ability to form flexible size lipid-nanodiscs has been well utilized in the structural studies of membrane-bound protein-protein complexes and to demonstrate the formation of lipid domains, mechanistic insights into these processes have been unknown. Here, for the first time, the dynamic nature of peptide-based lipid-nanodiscs is investigated by high speed atomic force microscopy (HS-AFM), which is capable of acquiring several images per second. 31P NMR, SEC and DLS were also used to monitor the association of lipid nanodiscs. The real time monitoring of nanodiscs in unprecedented time and space resolu-tions provides evidence for nanodisc association and the time scale of their fusion. This study provides the first experimental real-time direct evidence for the n...

Formation of pH‐Resistant Monodispersed Polymer–Lipid Nanodiscs

AbstractPolymer lipid nanodiscs are an invaluable system for structural and functional studies of membrane proteins in their near‐native environment. Despite the recent advances in the development and usage of polymer lipid nanodisc systems, lack of control over size and poor tolerance to pH and divalent metal ions are major limitations for further applications. A facile modification of a low‐molecular‐weight styrene maleic acid copolymer is demonstrated to form monodispersed lipid bilayer nanodiscs that show ultra‐stability towards divalent metal ion concentration over a pH range of 2.5 to 10. The macro‐nanodiscs (&gt;20 nm diameter) show magnetic alignment properties that can be exploited for high‐resolution structural studies of membrane proteins and amyloid proteins using solid‐state NMR techniques. The new polymer, SMA‐QA, nanodisc is a robust membrane mimetic tool that offers significant advantages over currently reported nanodisc systems.

Formation of pH‐Resistant Monodispersed Polymer–Lipid Nanodiscs

Polymer lipid nanodiscs are an invaluable system for structural and functional studies of membrane proteins in their near-native environment. Despite the recent advances in the development and usage of polymer lipid nanodisc systems, lack of control over size and poor tolerance to pH and divalent metal ions are major limitations for further applications. A facile modification of a low-molecular-weight styrene maleic acid copolymer is demonstrated to form monodispersed lipid bilayer nanodiscs that show ultra-stability towards divalent metal ion concentration over a pH range of 2.5 to 10. The macro-nanodiscs (>20 nm diameter) show magnetic alignment properties that can be exploited for high-resolution structural studies of membrane proteins and amyloid proteins using solid-state NMR techniques. The new polymer, SMA-QA, nanodisc is a robust membrane mimetic tool that offers significant advantages over currently reported nanodisc systems.